J. Piskorska-Pliszczynska

The cytosolic receptor binding affinities and ahh induction potencies of 29 polynuclear aromatic hydrocarbons

The dose-response rat hepatic cytosolic receptor-binding avidities, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction potencies in rat hepatoma H-4-II E cells in culture were determined for 29 polynuclear aromatic hydrocarbons. It was apparent that the magnitude of the EC50 values for these in vitro responses were strongly dependent on structure. Dibenz[a,h]anthracene (1.6 X 10(-8) M), 7-methylbenz[a]anthracene (1.6 X 10(-8) M), 3-methylcholanthrene (2.8 X 10(-8) M) and picene (4.5 X 10(-8) m) exhibited the highest affinity for the receptor protein and these compounds were only 5-fold less active the 2,3,7,8-tetrachlorodibenzo-p-dioxin (1 X 10(-8) M). All of the compounds which were active in the receptor-binding and monooxygenase enzyme-induction assays possessed one common structural feature, namely the presence of a phenanthrene structure fused with at least 1 benzo ring. The results also demonstrated that there was not any apparent correlation between the receptor-binding avidities and in vitro monooxygenase enzyme-induction potencies for the most active polynuclear aromatic hydrocarbons.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic and uterine estrogen receptor levels in rats

Administration of a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 20 or 80 micrograms/kg) resulted in significantly decreased hepatic and uterine estrogen receptor (ER) levels in 25-day-old female Long-Evans rats. By contrast, estradiol (5 and 15 micrograms/kg) administration increased hepatic and uterine ER levels, while a combination of 2,3,7,8-TCDD plus estradiol resulted in uterine and hepatic ER levels which were similar or lower than those observed after treatment with only 2,3,7,8-TCDD. In addition, 2,3,7,8-TCDD significantly decreased the effects of estradiol on uterine wet weight increase. Competitive binding studies showed that estradiol did not bind to the aryl hydrocarbon (Ah) receptor and that 2,3,7,8-TCDD did not bind to the ER. The effects of structure on the activity of polychlorinated dibenzo-p-dioxin congeners on their activity to down-regulate hepatic and uterine ER levels were determined by using 2,3,7,8-TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,3,7,8-TCDD, and 1,2,4,7,8-PeCDD. Both 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD exhibit high affinities for the Ah receptor and at dose levels of 80 micrograms/kg the hepatic ER levels were decreased 42 and 41%, respectively, and uterine ER levels were decreased 53 and 49%, respectively. 1,3,7,8-TCDD and 1,2,4,7,8-PeCDD bind less avidly to the Ah receptor and at dose levels of 400 micrograms/kg these compounds decreased hepatic ER levels 36 and 40%, respectively, and uterine ER levels 21 and 24%, respectively. These results support a role for the Ah receptor in the down-regulation of uterine and hepatic ER levels in the female rat by 2,3,7,8-TCDD and this effect may be associated with the decrease in spontaneous mammary and uterine tumors observed in female rats treated with 2,3,7,8-TCDD.

Polychlorinated dibenzofurans (PCDFs): Correlation between in vivo and in vitro structure-activity relationships

Fifteen polychlorinated dibenzofuran (PCDF) congeners were administered in a dose-response fashion to immature male Wistar rats and ED50 values for body weight loss, thymic atrophy and the induction of the hepatic microsomal cytochrome P-448-dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and 4-chlorobiphenyl hydroxylase were determined. There was an excellent correlation between the in vivo quantitative structure-activity relationships for these PCDFs and their in vitro activities as AHH inducers in rat hepatoma H-4-II E cells and as ligands for the 2,3,7,8-TCDD receptor protein. A comparison of isomers which differ at all 4 positions in the dibenzofuran ring system indicated that chlorine substitution at each position contributed differentially to the overall molecular activity [C-3 (or C-7) greater than C-2 (or C-8) greater than C-4 (or C-6) greater than C-1 (or C-9)]. There was also an excellent linear correlation between a plot of the -log ED50 for body weight loss vs. -log EC50 for in vitro AHH induction (correlation coefficient, r = 0.96) and -log ED50 for thymic atrophy vs. -log EC50 for in vitro AHH induction (correlation coefficient, r = 0.88). Since body weight loss and thymic atrophy in the rat are representative toxic responses to PCDFs and related toxic halogenated aryl hydrocarbons, the correlations noted above support the use of the in vitro AHH induction assay as a short term quantitative test system for this class of toxic halogenated aryl hydrocarbons.

Polychlorinated dibenzo-p-dioxins: quantitative in vitro and in vivo structure-activity relationships.

There were marked effects of structure on the activities of 14 polychlorinated dibenzo-p-dioxins (PCDDs) as competitive ligands for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor and as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells in culture. 2,3,7,8-TCDD was the most active compound in both assays and several PCDD congeners which were fully substituted in the lateral 2, 3, 7 and 8 positions but also contained additional chlorosubstituents in non-lateral 1, 4, 6 and 9 positions were less active. It was also evident that there was a decrease in in vitro binding and induction activities with decreasing lateral chlorine substitution. Although comparable structure-activity relationships (SARs) for the PCDDs were observed for the induction and receptor binding assays, there was not a linear or rank order correlation between the 2 sets of data. Several in vivo biologic and toxic activities of 2,3,7-trichloro-, 2,3,7,8- and 1,3,7,8-tetrachloro-, 1,2,4,7,8- and 1,2,3,7,8-pentachloro- and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin were determined in a dose-response fashion in immature male Wistar rats. The ED50 values for hepatic microsomal AHH and EROD induction, body weight loss and thymic atrophy were obtained. There was an excellent linear correlation between the -log EC50 values for AHH or EROD induction in cell culture and the -log ED50 values for enzyme induction, body weight loss and thymic atrophy in the rat. The in vitro enzyme induction data could be used to quantitatively estimate the toxicity of the PCDD congeners in the rat: this latter correlation has previously been observed for a series of polychlorinated dibenzofurans.

Polychlorinated dibenzofurans as 2,3,7,8-tcdd antagonists: In vitro inhibition of monooxygenase enzyme induction

2,4,6,8- and 1,3,6,8-tetrachlorodibenzofuran (TCDF) competitively displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the rat cytosolic receptor protein and their EC50 values were 1.5 X 10(-6) and 1.25 X 10(-7) M, respectively. In contrast to their relatively high binding avidities these TCDF isomers were poor inducers of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase in rat hepatoma H-4-II E cells in culture (EC50 greater than 10(-5) M). Coadministration of different concentrations of 2,4,6,8- and 1,3,6,8-TCDF (10(-5), 10(-6) and 10(-7) M) with 2 X 10(-10) M, 2,3,7,8-TCDD (a dose which elicits 80% of the maximal induction response) resulted in significant decreases in the expected (additive) induction of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by the mixture. Thus the partial agonists, 1,3,6,8- and 2,4,6,8-TCDF, antagonize the receptor-mediated enzyme induction activity of 2,3,7,8-TCDD presumably via competitive displacement of 2,3,7,8-TCDD from the receptor protein. In contrast, coadministration of 2,3,7,8-TCDF and 2,3,7,8-TCDD gave additive enzyme induction responses. The identification of the 2,3,7,8-TCDD antagonists represents a new class of halogenated aryl hydrocarbons.

The biologic and toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin in chickens

The administration of 2,3,7,8-TCDD to 2-week-old White Leghorn cockerels produced a dose-dependent induction of hepatic microsomal ethoxyresorufin O-deethylase (EROD) and benzo[alpha]pyrene (B[alpha]P) hydroxylase activities with induction EC50 values of 778 and 302 ng/kg, respectively. In addition, 2,3,7,8-TCDD also induced 4-dimethylaminoantipyrine (DMAP) N-demethylase (EC50 = 561 ng/kg), this result contrasting with studies reported for other animal species in which 2,3,7,8-TCDD either does not induce or inhibits this cytochrome P-450 dependent monooxygenase enzyme activity. However, the reduced cytochrome P-450:CO binding difference spectral absorption maxima for the 2,3,7,8-TCDD induced microsomes was observed at 448 nm which was similar to that reported for most animals which have been investigated. Electrophoresis of control and 2,3,7,8-TCDD induced microsomal proteins using SDS polyacrylamide slab gels showed intensification of 3 protein staining bands at Mr 53 000, 56 000 and 58 000. Incubation of the 2,3,7,8-TCDD-induced microsomes with SKF-525A and alpha-naphthoflavone showed that both compounds inhibited DMAP N-demethylase, EROD and AHH and that alpha-naphthoflavone was the more potent inhibitor of all 3 microsomal monooxygenases. 2,3,7,8-TCDD treatment caused significant involution of the Bursa of Fabricius when administered at a dose level of 10 micrograms/kg for 3 days and this result confirmed the extreme sensitivity of the immature White Leghorn cockerel to the biologic and toxic effects elicited by 2,3,7,8-TCDD. However, in contrast to other sensitive species, no high affinity cytosolic receptor protein for [3H]2,3,7,8-TCDD could be detected in the liver of chick embryos or 2-week-old birds.

Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: Characterization of 6-[125I]methyl-8-iodo-1, 3-dichlorodibenzofuran-Ah receptor complexes☆

6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.

Structure-dependent induction of aryl hydrocarbon hydroxylase activity in C57BL6 mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related congeners: Mechanistic studies

The time- and dose-dependent induction of murine hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities by five polychlorinated dibenzo-p-dioxin and dibenzofuran congeners showed that the order of induction potency was 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) greater than 2,3,7,8-tetrachlorodibenzofuran (TCDF) greater than 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD) greater than 1,2,3,7,8-pentachlorodibenzofuran (PCDF) greater than 2,3,7-trichlorodibenzo-p-dioxin (TrCDD). These structure-induction relationships were comparable to the structure-toxicity and competitive structure-receptor binding relationships previously reported for these compounds. However, using the corresponding radiolabeled congeners, the direct binding Kd values for dissociation of the cytosolic receptor-ligand complexes were 9.52, 7.96, 1.27, 3.10, and 8.31 nM for the 2,3,7,8-TCDD, 2,3,7,8-TCDF, 2,3,7-TrCDD, 1,2,3,7,8-PCDD, and 1,2,3,7,8-PCDF congeners and these data were clearly not structure dependent (i.e., similar to the structure-activity relationships). Some of the molecular properties for several radioligand-receptor complexes were similar; for example, the sedimentation coefficients for the cytosolic and nuclear receptor complexes varied from 8.8-10.4 S and 5.98-7.0 S, respectively, and the nuclear receptor complexes for all the radioligands eluted from a DNA-Sepharose column at salt concentrations of 0.27-0.29 M. Treatment of the mice with a maximum inducing dose of 2,3,7,8-[3H]TCDD resulted in a time-dependent formation of the nuclear receptor complex which was maximized between 16-24 hr and subsequently decreased up to 72 hr after initial exposure. In parallel studies, the nuclear receptor complex levels were determined 16 hr after treatment of the mice with different doses (2.25, 4.5, and 45 micrograms/kg) of all five radioligands. The results showed that at submaximal induction of the monooxygenase enzyme activities there was a linear correlation between the induced AHH or EROD activities (after 32 hr) and the corresponding nuclear receptor complex levels. It was also apparent from the data that the relative levels of nuclear receptor complex were structure dependent and this suggests that the transformation or activation of cytosolic receptor complexes may be a ligand structure-dependent process which correlates with the observed structure-activity relationships for 2,3,7,8-TCDD and related compounds.

Validation of in vitro bioassays for 2,3,7,8-TCDD equivalents

Abstract The inducibility of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells culture has been utilized as a quantitative bioassay for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). There was an excellent linear correlation between the -log EC50 (in vitro enzyme induction) for 16 PCDD and PCDF congeners and their -log ED50 values for in vivo toxicity (body weight loss and thymic atrophy) in the rat. There was also a comparable linear correlation between the in vitro and in vivo AHH/EROD induction activities. Extraction of a fly ash sample with methylene chloride followed by cleanup gave a PCDD/PCDF fraction which contained a complex mixture of isomers and congeners. The total PCDD and PCDF levels were 3.83 and 5.52 ug/g fly ash respectively and 2,3,7,8-TCDD constituted 2–3% of the total tetrachlorodibenzo-p-dioxins. Using the in vitro enzyme induction bioassay procedure, the estimated 2,3,7,8-TCDD (equivalents) in this extract were 105 ng/g fly ash. The doseresponse in vivo induction of AHH was also determined and the estimated 2,3,7,8-TCDD (equivalents) in this extract were 75 ng/kg fly ash and this confirms the excellent correlation between the in vivo and in vitro bioassays.

Development and validation of in vitro bioassays for 2,3,7,8-TCDD equivalents

Abstract Toxic halogenated aryl hydrocarbons are industrial compounds and/or by-products which have been detected as complex mixtures in almost every component of the global ecosystem. Included in this class of chemical pollutants are the halogenated (Cl/Br) biphenyls (PCBs/PBBs), dibenzo-p-dioxins (PCDDs/PBDDs) and dibenzofurans (PCDFs/PBDFs). Rat hepatoma H-4-II E cells are highly sensitive to the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-TCDD and related toxic halogenated aromatics and for > 25 of these compounds there was an excellent correlation (r = 0.85 to 0.93) between their in vitro pEC 50 values for induction and their pED 50 values for in vivo effects in the rat (AHH induction, body weight loss and thymic atrophy). Moreover, for selected PCB, PCDD and PCDF congeners comparable linear correlations between in vitro induction activity and in vivo toxicities in the guinea pig (AHH induction and body weight loss) and mouse (immunotoxicity) were also observed. The data confirm the utility of the in vitro induction bioassay for toxic halogenated aromatics. The results of these studies permit the calculation of estimated “2,3,7,8-TCDD equivalent ranges” for most of the important toxic halogenated aromatics and provide a more rational basis for risk assessment of these compounds and their mixtures.